Hemodialysis and hemofiltration membranes based upon a two-dimensional membrane material and methods employing same

ABSTRACT

Perforated graphene and other perforated two-dimensional materials can be used in hemodialysis membranes and blood filtration membranes for selective removal of components from blood in vivo and ex vivo. The membranes are useful in hemodialysis and hemofiltration techniques to provide improved patient care. Hemodialysis systems can include a hemodialysis membrane formed from perforated graphene or another perforated two-dimensional material disposed upon a porous support structure. Hemofiltration systems can include one or more and preferably two or more blood filtration membrane formed from perforated graphene or another perforated two-dimensional material disposed upon a porous support structure. Methods for performing hemodialysis can involve exposing blood from a patient to a hemodialysis membrane formed from a perforated two-dimensional material. Ex vivo dialysis techniques can be performed similarly. Methods for filtration of blood can involve passing blood through one or more filter membranes or through a plurality of sequential filter membranes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application62/044,877, filed Sep. 2, 2014, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD

The present disclosure generally relates to the use of nanomaterials inmedical applications, and, more specifically, to hemodialysis andhemofiltration membranes based upon graphene-based materials and othertwo-dimensional materials.

BACKGROUND

Hemodialysis is one of the most common treatments provided in medicalfacilities today, and the market for such treatments continues to grow.The 2013 global dialysis market is valued at $61.60 billion and isexpected to grow at a compound annual growth rate (CAGR) of 6.2% overthe next five years with the increasing number of end stage renaldisease (ESRD) patients and the rising prevalence of diabetes andhypertension worldwide. In addition, growth in the number of dialysisfacilities in developed as well as developing markets, increasingprivate investments, and venture funding to support new productdevelopment is contributing to the growth of the global market. Reducedinsurance disbursements to dialysis centers, high treatment costs, andlow awareness of kidney related diseases and their treatment modalitiesare factors that continue to restrain market growth.

Hemofiltration is typically employed with patients exhibiting acutekidney injury. In hemofiltration, water and relatively low molecularweight components (up to 20-30 kDa) are removed by convection through ahemofiltration membrane. Water and electrolytes are replaced in thepatient. Hemofiltration may be combined with hemodialysis.

FIG. 1 shows an illustrative schematic of a conventional hemodialysissystem and technique. In the illustrated system blood is passed from thepatient via an appropriate conduit (11) by action of pump (3) into adialyzer unit (5) which contains an appropriate filter (2), typically ahallow fiber filter, to selectively remove toxic species from the blood.Fresh dialysate is passed, employing pump (7), into the dialyzer viaappropriate conduit (14) and used dialysate exits the dialyzer unit viaappropriate conduit (12). A dialysate source (22) and waste receptacle(21) are optionally provided. Cleaned blood is returned to the patientvia appropriate conduit (13) through an air detector and trap (9). Inflow pressure into the dialyzer, venous pressure and arterial pressureare monitored (4, 6 and 8, respectively). A source of saline (16) andheparin (17) are provided via saline conduit (15) as needed via valvesor related fluid metering devices (18 and 19) to prevent clotting. FIG.2 shows an expanded schematic of a conventional hemodialysis membrane(30), having pores (32) of selected dimension to allow passage of ions(33), small molecules (34) and prevent passage of larger macromolecules(35). The thickness (t) of the convention membrane is in the range of 50micron. Current state solutions, or dialyzers, are hollow fiber membrane(30) devices in a hard plastic shell. Blood flows through the lumen ofthe fiber and dialysate flows through the dialyzer on the exterior ofthe fibers. Fibers are traditionally made of porous materials such ascellulose triacetate, polysulfone, polyethersulfone,polymethylmethacrylate, polyester polymer alloy, ethylene vinyl alcoholcopolymer or polyacrylonitrile. The fibers have a microporous structurethat allows small molecules to diffuse from the blood into thedialysate. The diffusion rate can be expressed in terms of the dialyzerclearance of the molecules. Clearances of various molecules can occur atdifferent rates under various blood and dialysate flow rate conditions.The large variety of dialyzer configurations permits physicians toappropriately specify a hemodialysis treatment to meet the needs of apatient. There is an entire system built around this filter technologyto provide the current standard of care to patients. However, theperformance is limited by the permeability, selectivity and roughness ofthe dialyzer membrane.

In view of the foregoing, improved hemodialysis membranes andhemofiltration membranes and methods would be of considerable benefit inthe art. In particular, hemodialysis membranes and hemofiltrationmembranes having increased permeability and selectivity would beespecially advantageous. The present disclosure satisfies the foregoingneeds and provides related advantages as well.

SUMMARY

The present disclosure describes membranes comprising a perforatedtwo-dimensional material disposed upon a porous support structure foruse in blood dialysis and blood filtration applications. Suchtwo-dimensional materials are selectively perforated to provide forselective removal of one or more components from the blood.Two-dimensional materials include for example graphene-based materials.

In one aspect the disclosure describes hemodialysis membranes andsystems in which perforated graphene-based material and other perforatedtwo-dimensional materials are used as a replacement for polymermembranes in conventional hemodialysis systems. The perforatedtwo-dimensional material, such as graphene-based material and graphene,can have a pore size of similar magnitude to those used in conventionalmembranes, while providing much greater permeability due the thinness ofthe graphene. In addition, the pores or perforations in thetwo-dimensional material, such as graphene-based material, can beselectively sized, functionalized, or otherwise manipulated to tailorthe selectivity of the hemodialysis separation process.

The present disclosure also describes hemodialysis methods in whichblood is exposed to a hemodialysis membrane formed from perforatedtwo-dimensional material, such as graphene-based material, and at leastone component is removed from the blood upon contacting the perforatedgraphene. In an embodiment, the at least on component removed is urea,measurement of the extent of removal of which can be used to assess theeffectiveness of a given hemodialysis method to remove low molecularweight toxic species, e.g., low molecular weight toxic species whichcontribute to disease. In an embodiment, at least one undesirablecomponent is removed, such as a low molecular weight toxic species or alower molecular weight (e.g., less than about 35 kDa) protein whichcontributes to uremia or other disorder, without removal of albumin atlevels detrimental to a given patient. In an embodiment, at least ureais removed without removal of detrimental levels of albumin.

The foregoing has outlined rather broadly the features of the presentdisclosure in order that the detailed description that follows can bebetter understood. Additional features and advantages of the disclosurewill be described hereinafter. These and other advantages and featureswill become more apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure, and theadvantages thereof, reference is now made to the following descriptionsto be taken in conjunction with the accompanying drawings describingspecific embodiments of the disclosure, wherein:

FIG. 1 shows an illustrative schematic of a conventional hemodialysissystem and technique;

FIG. 2 shows an expanded schematic of a conventional hemodialysismembrane. In this schematic a dialysis membrane is illustrated to havepores of selected dimension (diameter of for example about 2.4 nm) whichallows passage of ions, such as Na⁺; small molecules, such as urea, butdoes not allow passage of globular macromolecules, such as serumalbumin. Conventional hemodialysis membranes have thickness (t) in therange of 50 micron

FIG. 3 shows an illustrative scanning electron microscope (SEM) image ofperforated single-layer graphene-based material on a track-etchedpolycarbonate support structure;

FIGS. 4A-E show images of single layer graphene (nominal thickness ofabout 0.3 nm) and pores therein. FIG. 4A is a scanning transmissionelectron microscope (STEM) image of single-layer graphene with pores ofabout 1 nm. FIG. 4B is a micrograph of CDV graphene-based materialexhibiting pores ranging from about 0.5 to 1 nm in dimension. FIG. 4C isa micrograph of CDV graphene-based material exhibiting pores rangingfrom 2.5 to 7 nm in dimension. FIG. 4D is a micrograph of CDVgraphene-based material exhibiting a mean pore size dimension of 6.3 nm,and which is about 4% open with about 1×10¹¹ pores/cm². Perforations aregenerated in the CDV graphene of FIGS. 4A-D using ion beam irradiation.FIG. 4E is a micrograph of CDV graphene-based material in which poreswere introduced using focused ion beam (FIB) drilling and where theaverage pores size is 20 nm.

FIG. 5 shows an illustrative hemodialysis system containing agraphene-based membrane (55) and a two-chamber cross-flow vessel (51).This figure also illustrates an optional multiple pass hemodialysisconfiguration, implemented via optional conduit (65) in which useddialysate is mixed with fresh dialysate to decrease water use.

FIG. 6 shows an illustrative blood filtration configuration comprisingtwo or more (6 are shown) graphene-based membranes (71A-71F) each ofwhich have different pore dimensions, for example, where average poredimension increases from 71A to 71F. Passage of blood through the filterconfiguration generates two or more flow streams (6 are shown, 72A-72F)containing size separated components dependent upon the pore dimensionsof the filters. For example, where average pore dimension increases from71A to 71F, the flow streams from 72A-72F will contain components ofdecreasing size.

DETAILED DESCRIPTION

The present disclosure is directed to membranes comprising a perforatedtwo-dimensional material disposed upon a porous support structure foruse in blood dialysis and blood filtration applications. Suchtwo-dimensional materials are selectively perforated to provide forselective removal of one or more components from the blood. In specificembodiments, such two-dimensional materials are selectively perforatedto provide for selective removal of one or more selected undesirablecomponents from blood while retaining one or more selected desirablecomponents in the blood.

The present disclosure is directed, in part, to hemodialysis membranesand hemodialysis systems containing selectively perforated graphene oranother selectively perforated two-dimensional material. The presentdisclosure is also directed, in part, to methods for performing ahemodialysis treatment using a hemodialysis system containing suchperforated graphene or another such perforated two-dimensional material.Methods herein include hemodialysis cross flow configurations. Methodsherein include single-pass methods in which used dialysate is notrecirculated and multi-pass systems in which used dialysate is mixedwith fresh dialysate and reused.

The present disclosure is directed, in part, to blood filtrationmembranes and blood filtration systems containing selectively perforatedgraphene or another selectively perforated two-dimensional material. Thepresent disclosure is also directed, in part, to methods for performinga hemodialysis treatment using a hemodialysis system containing suchperforated graphene or another such perforated two-dimensional material

Graphene has garnered widespread interest for use in a number ofapplications due to its favorable mechanical and electronic properties.Graphene represents an atomically thin layer of carbon (or few carbonlayers) in which the carbon atoms reside as closely spaced atoms atregular lattice positions. The regular lattice positions can have aplurality of defects present therein, which can occur natively or beintentionally introduced to the graphene basal plane. Such defects willalso be equivalently referred to herein as “apertures,” “perforations,”or “holes.” The term “perforated graphene” will be used herein to denotea graphene sheet with defects in its basal plane, regardless of whetherthe defects are natively present or intentionally produced. Aside fromsuch apertures, graphene and other two-dimensional materials (e.g.,graphene oxide and the like) can represent an impermeable layer to manysubstances. Therefore, if they are sized properly, the apertures in theimpermeable layer can be useful in retaining entities that are largerthan the effective pore size. In this regard, a number of techniqueshave been developed for introducing a plurality of perforations ingraphene and other two-dimensional materials, where the perforationshave a desired size, number and chemistry about the perimeter of theperforations. Chemical modification of the apertures can allow entitieshaving particular chemical characteristics to be preferentially retainedor rejected as well.

Two-dimensional materials are, most generally, those which areatomically thin, with thickness from single-layer sub-nanometerthickness to a few nanometers, and which generally have a high surfacearea. Two-dimensional materials include metal chalogenides (e.g.,transition metal dichalogenides), transition metal oxides, hexagonalboron nitride, graphene, silicene and germanene (see: Xu et al. (2013)“Graphene-like Two-Dimensional Materials) Chemical Reviews113:3766-3798). Graphene represents a form of carbon in which the carbonatoms reside within a single atomically thin sheet or a few layeredsheets (e.g., about 20 or less) of fused six-membered rings forming anextended sp²-hybridized carbon planar lattice. In its various forms,graphene has garnered widespread interest for use in a number ofapplications, primarily due to its favorable combination of highelectrical and thermal conductivity values, good in-plane mechanicalstrength, and unique optical and electronic properties. Othertwo-dimensional materials having a thickness of a few nanometers or lessand an extended planar lattice are also of interest for variousapplications. In an embodiment, a two dimensional material has athickness of 0.3 to 1.2 nm. In other embodiments, a two dimensionalmaterial has a thickness of 0.3 to 3 nm.

In various embodiments, the two-dimensional material comprises a sheetof a graphene-based material. In an embodiment, the sheet ofgraphene-based material is a sheet of single- or multi-layer graphene ora sheet comprising a plurality of interconnected single- or multi-layergraphene domains. In embodiments, the multilayer graphene domains have 2to 5 layers or 2 to 10 layers. In an embodiment, the layer comprisingthe sheet of graphene-based material further comprises non-grapheniccarbon-based material located on the surface of the sheet ofgraphene-based material. In an embodiment, the amount of non-grapheniccarbon-based material is less than the amount of graphene. Inembodiments, the amount of graphene in the graphene-based material isfrom 60% to 95% or from 75% to 100%.

The technique used for forming the graphene or graphene-based materialin the embodiments described herein is not believed to be particularlylimited. For example, in some embodiments CVD graphene or graphene-basedmaterial can be used. In various embodiments, the CVD graphene orgraphene-based material can be liberated from its growth substrate(e.g., Cu) and transferred to a polymer backing. In some embodiments,the growth substrate may be corrugated before or after the graphenedeposition process to produce a graphene or graphene-based material withhigh surface area. In some embodiments, a growth substrate may be formedas a cylinder to form a sleeve of graphene or graphene-based material,thereby reducing the number of seams that must be sealed to form theenclosure.

The present inventors recognized that many of the techniques used tointroduce perforations into graphene-based material and othertwo-dimensional materials produce perforations having pore sizes similarto those present in conventional hemodialysis membranes. Thus, they canbe used for separating impurities having comparable size to thoseseparated using conventional hemodialysis membranes. However, sincesingle-layer or even multi-layer graphene are much thinner thanconventional hemodialysis membranes, a much greater transfer rate can berealized, as expressed with the following formula.

$\begin{matrix}{{{{Impurity}\mspace{14mu}{Transport}}{Q = {\frac{{Kd}^{2}A\; ɛ}{t}\left( {{\Delta\; P} - {{RT}\;\Delta\; C}} \right)}}Q = {{Impurity}\mspace{14mu}{flow}\mspace{14mu}\left( {{ml}\text{/}\sec} \right)}}{K = {{Membrane}\mspace{14mu}{{Permeability}/{pore}}\mspace{14mu}{area}}}{A = {{Membrane}\mspace{14mu}{Area}\mspace{14mu}\left( {m\; 2} \right)}}{ɛ = {porosity}}{{\Delta\; P} = {{transmembrane}\mspace{14mu}{pressure}\mspace{14mu}({Pa})}}{{\Delta\; C} = {{solute}\mspace{14mu}{transmembrane}\mspace{14mu}{concentration}}}{t = {{membrane}\mspace{14mu}{thickness}}}} & \lbrack 3\rbrack\end{matrix}$Hence, very thin graphene membranes allow for a much greater transportrate to be realized, which can be least 1000 times faster than inconventional hemodialysis membranes. In an embodiment, the graphenemembranes can be used as a drop-in replacement for conventional hemodialysis membranes.

In addition to the increased transport rate, the size selectivity canadvantageously allow decreased collateral metabolite loss to occurduring dialysis. Further, the smoothness of the graphene membrane canallow for a lower anticoagulant load to be used during a dialysisprocedure, and a reduced incidence of clotting can be realized. Finally,as a result of the foregoing, hemodialysis systems with a decreasedfootprint size and lower power requirements can be realized. Decreasedpatient treatment times can ultimately result. Any of these factors canincrease profitability of hemodialysis centers.

Likewise, the techniques for introducing perforations to the graphene orgraphene-based material are also not believed to be particularlylimited, other than being chosen to produce perforations within adesired size range. Perforations are sized as described herein toprovide desired selective permeability of a species (atom, molecule,protein, virus, cell, etc.) for a given application. Selectivepermeability relates to the propensity of a porous material or aperforated two-dimensional material to allow passage (or transport) ofone or more species more readily or faster than other species. Selectivepermeability allows separation of species which exhibit differentpassage or transport rates. In two-dimensional materials selectivepermeability correlates to the dimension or size (e.g., diameter) ofapertures and the relative effective size of the species. Selectivepermeability of the perforations in two-dimensional materials, such asgraphene-based materials, can also depend on functionalization ofperforations (if any) and the specific species that are to be separated.Separation of two or more species in a mixture includes a change in theratio(s) (weight or molar ratio) of the two or more species in themixture after passage of the mixture through a perforatedtwo-dimensional material.

Graphene-based materials include, but are not limited to, single layergraphene, multilayer graphene or interconnected single or multilayergraphene domains and combinations thereof. In an embodiment,graphene-based materials also include materials which have been formedby stacking single layer or multilayer graphene sheets. In embodiments,multilayer graphene includes 2 to 20 layers, 2 to 10 layers or 2 to Slayers. In embodiments, graphene is the dominant material in agraphene-based material. For example, a graphene-based materialcomprises at least 30% graphene, or at least 40% graphene, or at least50% graphene, or at least 60% graphene, or at least 70% graphene, or atleast 80% graphene, or at least 90% graphene, or at least 95% graphene.In embodiments, a graphene-based material comprises a range of grapheneselected from 30% to 95%, from 40% to 80%, from 50% to 70%, from 60% to95% or from 75% to 100%.

As used herein, a “domain” refers to a region of a material where atomsare uniformly ordered into a crystal lattice. A domain is uniform withinits boundaries, but different from a neighboring region. For example, asingle crystalline material has a single domain of ordered atoms. In anembodiment, at least some of the graphene domains are nanocrystals,having domain size from 1 to 100 nm or 10 to 100 nm. In an embodiment,at least some of the graphene domains have a domain size greater than100 nm to 1 micron, or from 200 nm to 800 nm, or from 300 nm to 500 nm.“Grain boundaries” formed by crystallographic defects at edges of eachdomain differentiate between neighboring crystal lattices. In someembodiments, a first crystal lattice may be rotated relative to a secondcrystal lattice, by rotation about an axis perpendicular to the plane ofa sheet, such that the two lattices differ in “crystal latticeorientation”.

In an embodiment, the sheet of graphene-based material comprises a sheetof single layer or multilayer graphene or a combination thereof. In anembodiment, the sheet of graphene-based material is a sheet of singlelayer or multilayer graphene or a combination thereof. In anotherembodiment, the sheet of graphene-based material is a sheet comprising aplurality of interconnected single or multilayer graphene domains. In anembodiment, the interconnected domains are covalently bonded together toform the sheet. When the domains in a sheet differ in crystal latticeorientation, the sheet is polycrystalline.

In embodiments, the thickness of the sheet of graphene-based material isfrom 0.34 to 10 nm, from 0.34 to 5 nm, or from 0.34 to 3 nm. In anembodiment, a sheet of graphene-based material comprises intrinsicdefects. Intrinsic defects are those resulting from preparation of thegraphene-based material in contrast to perforations which areselectively introduced into a sheet of graphene-based material or asheet of graphene. Such intrinsic defects include, but are not limitedto, lattice anomalies, pores, tears, cracks or wrinkles. Latticeanomalies can include, but are not limited to, carbon rings with otherthan 6 members (e.g. 5, 7 or 9 membered rings), vacancies, interstitialdefects (including incorporation of non-carbon atoms in the lattice),and grain boundaries.

In an embodiment, the layer comprising the sheet of graphene-basedmaterial further comprises non-graphenic carbon-based material locatedon the surface of the sheet of graphene-based material. In anembodiment, the non-graphenic carbon-based material does not possesslong range order and may be classified as amorphous. In embodiments, thenon-graphenic carbon-based material further comprises elements otherthan carbon and/or hydrocarbons. Non-carbon elements which may beincorporated in the non-graphenic carbon include, but are not limitedto, hydrogen, oxygen, silicon, copper and iron. In embodiments, thenon-graphenic carbon-based material comprises hydrocarbons. Inembodiments, carbon is the dominant material in non-grapheniccarbon-based material. For example, a non-graphenic carbon-basedmaterial comprises at least 30% carbon, or at least 40% carbon, or atleast 50% carbon, or at least 60% carbon, or at least 70% carbon, or atleast 80% carbon, or at least 90% carbon, or at least 95% carbon. Inembodiments, a non-graphenic carbon-based material comprises a range ofcarbon selected from 30% to 95%, or from 40% to 80%, or from 50% to 70%.

Such nanomaterials in which pores are intentionally created will bereferred to herein as “perforated graphene”, “perforated graphene-basedmaterials” or “perforated two-dimensional materials.” The presentdisclosure is also directed, in part, to perforated graphene, perforatedgraphene-based materials and other perforated two-dimensional materialscontaining a plurality of holes of size (or size range) appropriate fora given enclosure application. The size distribution of holes may benarrow, e.g., limited to a 1-10% deviation in size or a 1-20% deviationin size. In an embodiment, the characteristic dimension of the holes isselected for the application. For circular holes, the characteristicdimension is the diameter of the hole. In embodiments relevant tonon-circular pores, the characteristic dimension can be taken as thelargest distance spanning the hole, the smallest distance spanning thehole, the average of the largest and smallest distance spanning thehole, or an equivalent diameter based on the in-plane area of the pore.As used herein, perforated graphene-based materials include materials inwhich non-carbon atoms have been incorporated at the edges of the pores.

In various embodiments, the two-dimensional material comprises graphene,molybdenum disulfide, or boron nitride. In more particular embodiments,the two-dimensional material can be graphene. Graphene according to theembodiments of the present disclosure can include single-layer graphene,multi-layer graphene, or any combination thereof. Other nanomaterialshaving an extended two-dimensional molecular structure can alsoconstitute the two-dimensional material in the various embodiments ofthe present disclosure. For example, molybdenum sulfide is arepresentative chalcogenide having a two-dimensional molecularstructure, and other various chalcogenides can constitute thetwo-dimensional material in the embodiments of the present disclosure.Choice of a suitable two-dimensional material for a particularapplication can be determined by a number of factors, including thechemical and physical environment into which the graphene or othertwo-dimensional material is to be terminally deployed. For applicationin the present invention, materials employed in making an enclosure arepreferably biocompatible or can be made biocompatible.

The process of forming holes in graphene and other two-dimensionalmaterials will be referred to herein as “perforation,” and suchnanomaterials will be referred to herein as being “perforated.” In agraphene sheet an interstitial aperture is formed by each six-carbonatom ring structure in the sheet and this interstitial aperture is lessthan one nanometer across. In particular, this interstitial aperture isbelieved to be about 0.3 nanometers across its longest dimension (thecenter to center distance between carbon atoms being about 0.28 nm andthe aperture being somewhat smaller than this distance). Perforation ofsheets comprising two-dimensional network structures typically refers toformation of holes larger than the interstitial apertures in the networkstructure.

Due to the atomic-level thinness of graphene and other two-dimensionalmaterials, it can be possible to achieve high liquid throughput fluxesduring separation or filtration processes, even with holes that are inthe ranges of 1-200 nm, 1-100 nm, 1-50 nm, or 1-20 nm.

Chemical techniques can be used to create holes in graphene and othertwo-dimensional materials. Exposure of graphene or anothertwo-dimensional material to ozone or atmospheric pressure plasma (e.g.,an oxygen/argon or nitrogen/argon plasma) can effect perforation. Othertechniques, such as ion bombardment, can also be used to remove matterfrom the planar structure of two-dimensional materials in order tocreate holes. All such methods can be applied for preparation ofperforated two-dimensional materials for use herein dependent upon thehole sizes or range of hole sizes desired for a given application. Theterms holes, pores, apertures and perforations are used interchangeablyherein.

In various embodiments of the present disclosure, the holes produced inthe graphene-based material or other two-dimensional material can rangefrom about 0.3 nm to about 50 nm in size. In a more specific embodiment,hole sizes can range from 1 nm to 50 nm. In a more specific embodiment,hole sizes can range from 1 nm to 10 nm. In a more specific embodiment,hole sizes can range from 5 nm to 10 nm. In a more specific embodiment,hole sizes can range from 1 nm to 5 nm. In a more specific embodiment,the holes can range from about 0.5 nm to about 2.5 nm in size. In anadditional embodiment, the hole size is from 0.3 to 0.5 nm. In a furtherembodiment, the hole size is from 0.5 to 10 nm. In an additionalembodiment, the hole size is from 5 nm to 20 nm. In a furtherembodiment, the hole size is from 0.7 nm to 1.2 nm. In an additionalembodiment, the hole size is from 10 nm to 50 nm.

FIG. 3 shows an illustrative SEM image of perforated single-layergraphene on a track-etched polycarbonate support structure. Suchconfigurations can be used as a hemodialysis membrane in variousembodiments of the present disclosure. In general, any porous supportstructure that is suitably biocompatible with blood can be used as asupport for the perforated graphene in the various embodiments of themembranes described herein. FIG. 4A shows a high magnification STEMimage of the single-layer graphene and the pores therein. FIGS. 4B-4Dare micrographs of single-layer graphene exhibiting different poredimension ranges (or average pore dimension) and different poredensities. FIG. 4B illustrates CDV graphene-based material perforatedwith ion beam (Xe, 500V accelerating voltage, (60 nAs=3.75×1013ions/cm2), neutralizer used), while suspended with background gas (airat 1×10-4 Torr). FIG. 4C illustrates CVD graphene-based materialperforated with ion beam (Xe 500V, 60 nAs fluence (52 nA flux for 1.14sec), no neutralizer used) while suspended with background gas (air at1×10-4 Torr). FIG. 4D illustrates CDV graphene-based material perforatedwith ion beam (high-fluence (2000 nAs=1.25×1015 ions/cm2), low energy(20V accelerating voltage) Xe ions) while suspended.

Methods for perforating two-dimensional materials, includinggraphene-based materials and graphene have been described in the art andinclude among others, irradiation with ions, bombardment with particles,etching processes and focus ion beam drilling. Methods which allowingformation of pores or perforations of a selected size (dimension) arepreferred. Pores may have any useful shape and may be substantiallyround or may be elongated, e.g., slit-shaped. The terms size anddimension of a pore refer to the widest dimension of the pore whichdepend upon the shape of the pore. The widest dimension of a round poreis the diameter of the round pore. In preferred embodiments, poredimensions in dialysis membranes and filters range from about 1 nm toabout 30 nm, or from about 1 nm to about 20 nm, or from about 1 nm toabout 10 nm or from 1 nm to about 7 nm. In more specific embodiments,pores dimensions in the membranes and filters herein range up to 7 nm.

In an embodiment, the membranes herein are useful in filteringapplications where high sheer is applied to reduce fouling

FIG. 4E illustrates pores formed using focused ion beam drilling wherethe average pore dimension is 20 nm. Few-layer graphene (up to about 20graphene layers) can also be used in various embodiments of the presentdisclosure. Exemplary dimensions of the apertures in the graphene can beabout 30 nm or less in size, 20 nm or less in size 10 nm or less insize, 7 nm or less in size, 5 nm or less in size, about 2 nm or less insize, or about 1 nm or less in size.

In accordance with the disclosure, a perforated graphene membranemounted on a suitable bio-compatible support structure can beconfigured, for example, in a two chamber cross-flow vessel in a similarmanner to today's polymer hemodialysis membranes. FIG. 5 shows anillustrative hemodialysis system containing a graphene-based membranewithin a two-chamber cross-flow vessel. In this exemplary configuration(50), a two-chamber crossflow vessel (51) having a first chamber (52)for flow of draw solution (e.g., dialysate) and a second chamber (53)for flow of blood is provided with a selectively perforated membrane ofgraphene-based material (55). A planar or flat sheet membraneconfiguration is shown. It will be appreciated that alternative membraneconfigurations can be employed, such as spiral wound membraneconfiguration. In the membrane (55), the perforated graphene material issupported on a biocompatible porous polymer. The membrane isappropriately mounted and sealed within the vessel (51) employing anyconventional method that provides an appropriate leak-proof seal. Forexample, the membrane can be mounted between two biocompatible matingframes with appropriate biocompatible gaskets. Alternatively, themembrane can be mounted and sealed using a biocompatible adhesive.

In general, contaminated blood entering via conduit 56 moves across afirst surface of the graphene membrane (55), controlled transportchannels in its surface (nominally 1 atom thick and defined by the poresizes of the perforations) allow a high flow rate of contaminants to beremoved very efficiently from the blood and transported across themembrane into the other side of the chamber (52) where a suitable drawsolution (such as a dialysate) entering via conduit 58, solubilizes orentrains the contaminants and carries them away for disposal via conduit59. Cleaned blood exits the system via conduit 57 and as shown in FIG. 1can be returned to the patient via an intervening air trap. Multi-layerperforated graphene material as well as other two-dimensional materialscan be used in a similar manner. Dialysate is passed through the systememploying a pump (60). A blood pump (not shown) can be used (asillustrated in FIG. 1) for passage of blood through chamber 53. Flowpressure in the system can be monitored as illustrated in FIG. 1. Afresh dialysate receptacle (61) and a waste dialysate receptacle can beprovided (62).

In a related multi-pass configuration, used dialysate exiting viaconduit 59 can in whole or in part be transferred via conduit 65 to bemixed with fresh dialysate for recirculation through the system.Recirculation of dialysate decreases the volume of dialysate needed. Inan embodiment in a multi-pass configuration, the used dialysate, such asthat exiting via conduit 59 can be filtered using a membrane asdescribed herein having selected pore size to remove/reduce the levelsof undesired contaminant in the used dialysate.

It is known in the art of hemodialysis that it can be important toemploy dialysate with minimum undesired components. Thus filteringdevices employing membranes of this disclosure which comprisesselectively perforated two-dimensional materials, such as graphene, canalso be employed in the preparation of dialysate or be employed topre-filter dialysate prior to introduction into a dialyzer.

Alternate fluidic arrangements that optimize the transformationaltransport across the graphene membrane can also be used. Anotherembodiment with a sequence of concatenated filter chambers can alleviatethe need for a diffusively active draw solution.

Regardless of the utilized membrane configuration, as a direct result ofthe increased transport efficiency, the patient treatment time can begreatly reduced, the level of currently infused anti-coagulants (such asheparin) can be greatly reduced because of the graphene surfaceneutrality and smoothness (minimizing stirring and agitation that cantrigger the clotting sequence), and the rate of auxiliary metaboliteremoval can be carefully controlled so as to minimize depletion ofbeneficial electrolytes with simultaneous removal of undesiredcontaminants. Use of the membranes of this disclosure has the potentialto decrease complement activation which can lead to allergic reactionsduring treatment and may also lead to acute intradialytic pulmonaryhypertension, chronic low-grade systemic inflammation and leukocytedysfunction.

In some embodiments, the graphene or other two-dimensional material canbe functionalized. Particularly, the perimeter of the apertures withinthe graphene can be functionalized. Suitable techniques forfunctionalizing graphene will be familiar to one having ordinary skillin the art. Moreover, given the benefit of the present disclosure and anunderstanding consistent with one having ordinary skill in the art, askilled artisan will be able to choose a suitable functionality forproducing a desired interaction with an entity in a fluid, such as abiological fluid. For example, the apertures in a graphene can befunctionalized such that they interact preferentially with a protein orclass of proteins in deference to other biological entities of similarsize, thereby allowing separations based upon chemical characteristicsto take place. In some embodiments, pores of a given two-dimensionalmaterial are functionalized with a chemical species that is positivelycharge at physiologic pH (e. g., carries one or more amine groups). Insome embodiments, pores of a given two-dimensional material arefunctionalized with a chemical species that is positively negativelycharged at physiologic pH (e. g., carries one or more carboxyl orsulfonate groups). In some embodiments, pores of a given two-dimensionalmaterial are functionalized with a chemical species that is hydrophobicand in other embodiments pores of a given two-dimensional material arefunctionalized with a chemical species that is hydrophilic.

In some embodiments, the graphene or other two-dimensional material canbe functionalized with a chemical entity so that the functionalizationinteracts preferentially with a particular type of biological entity(e.g., by a chemical interaction). In some or other embodiments, thegraphene or other two-dimensional material can be functionalized suchthat it interacts electrically with a biological entity (e.g., by apreferential electrostatic interaction). Selective interactions basedupon biological recognition are also possible.

Membranes herein include a perforated two-dimensional material supportedon a porous substrate. The porous material is preferably biocompatibleand in some embodiments is preferably suitable for implantation in ahuman or animal body. The porous substrate can be a polymer, ceramic ormetal. Suitable materials include among others, poly(methylmethacrylate) (PMMA), polyesters, polyamides, polyimides, polyethylene,polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene fluoride(PVDF), polycarbonate, polyetherether-ketone polymers, i.e., PEEK™polymers (Trademark Victrex, USA, Inc.) and particularlypolyaryletheretherketone, polyvinyl chloride (PVC), and mixtures,copolymers and block copolymers thereof. Additionally, non-polymericsubstrates such as Si, SiN, anodized alumina, porous ceramics, orsintered metals can be employed. In specific embodiments, the substrateis a biocompatible polymer. In an embodiment, suitable polymers forforming a porous or permeable fibrous layer are biocompatible, bioinertand/or medical grade materials. In specific embodiments, the substrateis a track-etched polymer. In specific embodiments, the substrate istrack-etched polycarbonate.

In an embodiment, the support can itself have a porous structure whereinthe pores are larger than those of the two-dimensional material. In anembodiment, the support structure is entirely porous. In embodiments,the support structure is at least in part non-porous.

In embodiments herein the two-dimensional material is a graphene-basedmaterial. In embodiments of herein, the two-dimensional material isgraphene.

In embodiments herein at least a portion of the holes in thetwo-dimensional materials of the membranes are functionalized.

In embodiments herein at least a portion of the two-dimensional materialis conductive and a voltage can be applied to at least a portion of theconductive two-dimensional material. The voltage can be an AC or DCvoltage. The voltage can be applied from a source external to themembrane. In an embodiment, a membrane herein further comprisesconnectors and leads for application of a voltage from an externalsource to the two-dimensional material. Application of an electricalcharge to a conductive membrane herein can additionally facilitateselective or targeted removal of components from blood, dialysate,and/or water. Additionally, the conductive properties of graphene-basedor other two-dimensional membranes can allow for electrification to takeplace from an external source. In exemplary embodiments, an AC or DCvoltage can be applied to conductive two-dimensional materials of theenclosure. The conductivity properties of graphene-based materials andgraphene can provide additional gating to charged molecules.Electrification can occur permanently or only for a portion of the timeto affect gating. Directional gating of charged molecules can bedirected not only through the pores (or restrict travel through pores),but also to the surface of the graphene to adsorb or bind

Membranes herein can also be employed in blood filtration applications.In such applications, blood is passed through one or more or preferablytwo or more membranes in sequence to selectively remove components fromthe blood by size. For a membrane of given pore dimension, components ofsufficiently smaller dimension compared to the pores will pass throughthe pores of the membrane while components of sufficiently largerdimension compared to the pores will not. Thus, filtration of selectedblood components can be accomplished by passage of the blood through oneor more membranes with selected pore dimensions.

An exemplary filtration configuration is illustrated in FIG. 6. In thisconfiguration, blood is passed sequentially through a plurality ofmembranes at least two of which have different pore dimensions or poredensities. Preferably, at least two of the plurality of membranes havedifferent pore dimensions. In the illustrated embodiment, six membranesare provided (71A-71F). Preferably, each of the membranes in thefiltration configuration has different pore dimension. In a specificembodiment, the pore size dimension of the membranes decreases in thedirection of blood flow. Passage of blood sequentially through themembranes generates flows (72A-72F are shown) which contain bloodcomponents separated by size. The separated flows can be individuallycollected, individually discarded or two or more of the flows can becombined for any appropriate use.

As discussed above, hemodialysis and hemofiltration are employed toremove toxic substances such as creatinine and urea from the bloodtypically to replace or supplement such function of the kidneys. Theterm “removed” is used herein to encompass a decrease in level of thecomponent after dialysis or filtration. It is noted that the termremoved includes decreasing the level of toxic species in the blood tonon-toxic levels or to with the range of concentrations found in thosewhose individuals who have normal kidney function. During hemodialysisand hemofiltration, it is undesirable, as is known in the art, to removeor significantly lower the concentration of certain components belowtheir normal concentration range in individuals with normal kidneyfunction. One such component is serum albumin the removal of too much ofwhich can be detrimental to an individual. It is generally known in theart which blood components should be removed and which should beretained to in general achieve component levels that are within thenormal concentration level of the components in the blood. In somecases, hemodialysis and hemofiltration are performed continuously in anattempt to maintain levels of toxic species in the blood atconcentrations the same as those in individuals with normal kidneyfunction. In many cases however, hemodialysis and hemofiltration areperformed intermittently (e.g., on a set schedule) to lower levels oftoxic species in the blood to normal or below normal levels. During thetime between treatments, the levels of toxic species can build up in theblood.

The membranes of this disclosure formed by introduction of pores ofselective dimension into sheets or layers of two-dimensional materialare particularly suited to targeted removal of components based on size.As illustrated in FIGS. 4A-4E, methods are available in the art forintroduction of pores of different dimensions which allow for suchtargeted removal. For example, two-dimensional material having averagepore dimension or size of 20 nm will allow passage of water, ions andmost small molecules (molecular weight of 500 or less) and will alsoallow passage of many proteins. Two-dimensional materials having averagepore size of 7 nm will allow a passage of water, ions and most smallmolecules (molecular weight of 500 or less), but will not allow passageof many protein species, such as serum albumin. Two-dimensionalmaterials having average pore size of about 1 nm will allow passage ofwater and atomic ions generally, but will not allow passage of manymolecular components. Choice of pore dimensions in a given membraneallows targeted removal of components from a liquid, such a blood.

Although the disclosure provided herein is primarily directed tohemodialysis membranes and blood filtration membranes formed fromgraphene materials it is to be recognized that graphene oxide (GO) andreduced graphene oxide (rGO) can also be used in alternativeembodiments. It will be appreciated that filtration devices containingmembranes and membranes herein may be prepared from combinations oftwo-dimensional materials. Other perforated two-dimensional materialscan also be used as well. In addition to in vivo hemodialysis andhemofiltration techniques, ex vivo dialysis and filtration techniquesare also contemplated as well.

Methods for treating a patient using the disclosed membranes are alsocontemplated herein. These treatment methods are performed using thedisclosed membranes in a manner similar to that used with conventionalhemodialysis or hemofiltration techniques. In brief, the methods involvecontacting blood from a patient with a graphene-based hemodialysis orhemofiltration membrane (or membrane configuration, as illustrated inFIG. 6) in order to remove one or more contaminants therefrom.Contaminants removed from the blood by hemodialysis can then be removedin a dialysis fluid or those removed by filtration in a separated flowcan be removed or collected as desired. The purified blood can then berecirculated to the patient. In an embodiment, hemodialysis methodsherein are combined with blood filtration methods herein. In anembodiment, conventional hemodialysis methods herein are combined withblood filtration methods herein. Hemodialysis membranes and bloodfiltration membranes herein can also be employed in implantable devices,such as art-contemplated artificial kidneys and bioartifical kidneys.

The membranes herein can further be employed for peritoneal dialysis andin renal assist devices. Peritoneal dialysis is also employed to removewaste products from blood when normal kidney function is lost orimpaired. Blood vessels in the abdominal lining (the peritoneum) replacethe function of the kidneys when a dialysate is flowed into and out ofthe peritoneal space. Membranes herein can be employed for filtration ofdialysate in peritoneal dialysis. Renal assist devices include wearableand implantable devices for hemodialysis and peritoneal dialysis.Membranes herein can be employed to implement such devices as dialysismembranes and/or filtration devices. Certain renal assist devices (e.g.,bioartifical kidneys) include biological cells for carrying out certainmetabolic functions. For example, an implantable artificial kidney caninclude a bio-cartridge of renal tubule cells which, mimic the metabolicand water-balance function of the kidneys. Two-dimensional materials,particularly graphene-based materials can be employed as selectivelypermeable enclosures to retain such cells and to allow selective entryof components into the enclosure and selective exit of components fromthe enclosure. Such enclosures can for example be employed in artificialkidney which contain a bio-cartridge. Such enclosure are described forexample in U.S. application Ser. No. 14/656,190, filed, which isincorporated by reference herein in its entirety for descriptions ofsuch enclosures.

Ex vivo dialysis techniques can be conducted similarly. Such dialysistechniques can be conducted upon a biological fluid, such as blood, orupon other dialyzable fluids in need of contaminant removal therefrom.

Although the disclosure has been described with reference to thedisclosed embodiments, one having ordinary skill in the art will readilyappreciate that these are only illustrative of the disclosure. It shouldbe understood that various modifications can be made without departingfrom the spirit of the disclosure. The disclosure can be modified toincorporate any number of variations, alterations, substitutions orequivalent arrangements not heretofore described, but which arecommensurate with the spirit and scope of the disclosure. Additionally,while various embodiments of the invention have been described, it is tobe understood that aspects of the disclosure may include only some ofthe described embodiments. Accordingly, the disclosure is not to be seenas limited by the foregoing description.

Every formulation or combination of components described or exemplifiedcan be used to practice the invention, unless otherwise stated. Specificnames of compounds are intended to be exemplary, as it is known that oneof ordinary skill in the art can name the same compounds differently.When a compound is described herein such that a particular isomer orenantiomer of the compound is not specified, for example, in a formulaor in a chemical name, that description is intended to include eachisomer and enantiomer of the compound described individually or in anycombination. One of ordinary skill in the art will appreciate thatmethods, device elements, starting materials and synthetic methods otherthan those specifically exemplified can be employed in the practice ofthe invention without resort to undue experimentation. All art-knownfunctional equivalents, of any such methods, device elements, startingmaterials and synthetic methods are intended to be included in thisinvention. Whenever a range is given in the specification, for example,a temperature range, a time range, or a composition range, allintermediate ranges and subranges, as well as all individual valuesincluded in the ranges given are intended to be included in thedisclosure. When a Markush group or other grouping is used herein, allindividual members of the group and all combinations and subcombinationspossible of the group are intended to be individually included in thedisclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, limitation or limitations which is notspecifically disclosed herein.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The precedingdefinitions are provided to clarify their specific use in the context ofthe invention.

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art, insome cases as of their filing date, and it is intended that thisinformation can be employed herein, if needed, to exclude (for example,to disclaim) specific embodiments that are in the prior art. Forexample, when a compound is claimed, it should be understood thatcompounds known in the prior art, including certain compounds disclosedin the references disclosed herein (particularly in referenced patentdocuments), are not intended to be included in the claims.

What is claimed is the following:
 1. A medical device comprising: anenclosure comprising a blood filtration membrane, wherein the bloodfiltration membrane comprises a perforated two-dimensional materialcomprising perforated graphene, the perforated two-dimensional materialbeing disposed upon a porous support structure, and renal tubule cellsdisposed within the enclosure; wherein: the perforated graphene isconfigured to retain the renal tubule cells; and the perforated grapheneis configured to allow selective entry and/or exit of at least onecomponent contained in blood into and/or out of the enclosure.
 2. Themedical device of claim 1, wherein the perforated two-dimensionalmaterial is graphene-based material.
 3. The medical device of claim 2,wherein the graphene-based material is single-layer graphene.
 4. Themedical device of claim 1, wherein the perforated two-dimensionalmaterial is graphene oxide.
 5. A method comprising: exposing blood to ablood filtration device, the blood filtration device comprising: anenclosure comprising a blood filtration membrane, wherein the bloodfiltration membrane comprises a perforated two-dimensional materialcomprising perforated graphene, the perforated two-dimensional materialbeing disposed upon a porous support structure, and renal tubule cellsdisposed within the enclosure; wherein: the perforated graphene isconfigured to retain the renal tubule cells; and the perforated grapheneis configured to allow selective entry and/or exit of at least onecomponent contained in blood into and/or out of the enclosure.
 6. Themethod of claim 5, wherein the perforated two-dimensional material isgraphene-based material.
 7. The method of claim 6, wherein thegraphene-based material is single-layer graphene.
 8. The method of claim5, wherein the perforated two-dimensional material is graphene oxide. 9.The medical device of claim 1, wherein: the perforated graphene isconfigured to deny selective entry of at least one component containedin blood into the enclosure; and the at least one component denied entrycomprises one or more proteins.
 10. The method of claim 5, furthercomprising implanting the blood filtration membrane into a subject inneed thereof.
 11. The method of claim 10, wherein the subject is adiabetic subject.
 12. The method of claim 10, wherein the method is amethod of treating diabetes.
 13. The medical device of claim 1, whereinthe porous structure comprises a biocompatible polymer selected from thegroup of poly(methyl methacrylate) (PMMA), polyesters, polyamides,polyimides, polyethylene, polypropylene, polytetrafluoroethylene (PTFE),polyvinylidene fluoride (PVDF), polycarbonate, polyetherether-ketonepolymers, polyaryletheretherketone, polyvinyl chloride (PVC), andmixtures, copolymers or block copolymers thereof.
 14. The medical deviceof claim 1, wherein the at least one component contained in bloodcomprises a waste product.
 15. The medical device of claim 14, whereinthe waste product comprises urea.
 16. The medical device of claim 9,wherein the one or more proteins comprise albumin.
 17. The medicaldevice of claim 1, wherein the medical device is a bioartificial kidney.18. The method of claim 5, wherein the at least one component containedin blood comprises a waste product and the waste product comprises urea.19. The method of claim 5, wherein when the blood filtration membrane iscontacted with blood at least one component contained in the blood isdenied selective entry into the device; and the at least one componentdenied entry comprises one or more proteins.
 20. A blood filtrationdevice comprising: an enclosure comprising a blood filtration membrane,wherein the blood filtration membrane comprises at least one layer of aperforated two-dimensional graphene, the perforated two-dimensionalmaterial being disposed upon a porous support structure, and renaltubule cells disposed within the enclosure; wherein: the perforatedgraphene comprises apertures about 30 nm or less in size.